EP0807123A1 - Molecules interactives du recepteur du facteur de stimulation des colonies de granulocytes - Google Patents

Molecules interactives du recepteur du facteur de stimulation des colonies de granulocytes

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Publication number
EP0807123A1
EP0807123A1 EP95911668A EP95911668A EP0807123A1 EP 0807123 A1 EP0807123 A1 EP 0807123A1 EP 95911668 A EP95911668 A EP 95911668A EP 95911668 A EP95911668 A EP 95911668A EP 0807123 A1 EP0807123 A1 EP 0807123A1
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EP
European Patent Office
Prior art keywords
antibody
molecule
animal
csf
immunointeractive
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95911668A
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German (de)
English (en)
Other versions
EP0807123A4 (fr
Inventor
Judith Eleanor Layton
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ludwig Institute for Cancer Research Ltd
Ludwig Institute for Cancer Research New York
Original Assignee
Ludwig Institute for Cancer Research Ltd
Ludwig Institute for Cancer Research New York
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Filing date
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Application filed by Ludwig Institute for Cancer Research Ltd, Ludwig Institute for Cancer Research New York filed Critical Ludwig Institute for Cancer Research Ltd
Priority to EP01118103A priority Critical patent/EP1156063A1/fr
Publication of EP0807123A1 publication Critical patent/EP0807123A1/fr
Publication of EP0807123A4 publication Critical patent/EP0807123A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to cytokine receptor interactive molecules
  • the present invention provides immunoreactive molecules such as antibodies capable of interacting with granulocyte colony stimulating factor receptor.
  • G-CSF Granulocyte colony-stimulating factor stimulates the proliferation and 5 differentiation of neutrophil precursors via interaction with a specific cell surface receptor, the G-CSF receptor (G-CSF-R).
  • the G-CSF-R has been cloned (1) and is functionally active in several different cell types (2). However, little is known about the mechanism of signal transduction.
  • the G-CSF-R is believed to consist of a single chain that is activated 0 through ligand induced homodimerisation (3) as has been shown for the erythropoietin and growth hormone receptors (EPO-R, GH-R) (4).
  • the G-CSF-R does not contain an intrinsic protein kinase domain (1) although tyrosine kinase activity seems to be required for transduction of the G-CSF signal (5).
  • the present invention provides for the first time immune reactive molecules capable of interacting with G-CSF-R which will facilitate the development of new therapeutic and diagnostic molecules based on the immune reactive molecules or agonists or antagonists thereof.
  • One aspect of the present invention contemplates an immunointeractive molecule capable of binding or otherwise associating with an animal G-CSF receptor
  • the animal is a mammal such as a human or murine species.
  • the immunointeractive molecule is a polyclonal or monoclonal antibody.
  • Another aspect of the present invention is directed to a diagnostic agent comprising an immunointeractive molecule capable of binding or otherwise associating with an animal G-CSF-R extracellular domain.
  • the immunointeractive molecule is an antibody labelled with a reporter molecule.
  • Yet another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an immunointeractive molecule as contemplated above together with one or more pharmaceutically acceptable carriers and/or diluents.
  • Still another aspect of the present invention provides a method for treating a G-CSF associated disease condition such as a cancer or tumour in a mammal, said method comprising administering to said mammal an effective amount of an immunointeractive molecule capable of binding or otherwise associating with an animal G-CSF-R extracellular domain.
  • Figure 1 is a FACS analysis of anti-G-CSF-R antibody binding to CHO-6A11 cells (solid histogram) compared with control CHO-10 cells (open histograms). The graphs show relative cell number versus fluorescence intensity (log scale).
  • Figure 2 shows immunoprecipitation and Western blotting assay of some of the anti-G-CSF-R antibodies.
  • Left panel CHO-6A11 cell lysates were immunoprecipitated with the test monoclonals, the precipitates run on SDS-PAGE and blotted with R ⁇ G-CSF-R antibody.
  • Right panel CHO-6A11 cell lysates were immunoprecipitated with LMM741, the precipitates run on SDS-PAGE and blotted with the test monoclonals. Results obtained with the antibodies not shown are given in Table 2.
  • Figure 3 shows inhibition of FD.hGR3 proliferation by anti-G-CSF-R antibodies.
  • FD.hGR3 cells were cultured with G-CSF and antibody dilutions as described in the examples. The antibodies not shown did not cause inhibition, giving results that were similar to LMM791.
  • Figure 4 shows inhibition of BAF.hGR proliferation by anti-G-CSF-R antibodies.
  • BAF.hGR cells were cultured with G-CSF and antibody dilutions as described in the examples. Data from the antibodies not shown are given in Table 2.
  • Figure 5 shows inhibition of binding of 125 I-G-CSF to BAF.hGR cells by anti- G-CSF-R antibodies.
  • the antl odies not shown gave results that were similar to LMM847.
  • an immunointeractive molecule capable of binding or otherwise associating with an animal G-CSF-R, and in particular, the extracellular domain of such a receptor.
  • the immunointeractive molecules are in the form of antibodies such as polyclonal or monoclonal antibodies although monoclonal antibodies are preferred.
  • the present invention also extends to immunologically interactive fragments, parts, derivatives, homologues or analogues of these antibodies.
  • Such antibodies may be in isolated or purified form meaning that a composition comprises at least 25 % , more preferably at least 35%, even more preferably at least 45-50%, still more preferably at least 60-75% and even still more preferably at least 80-95% of the antibodies as determined by weight, immunoreactivity or other convenient means.
  • the antibodies may be present in the form of isolated culture supernatant, tissue extract, serum or whole blood or ascites fluid.
  • the animal G-CSF-R is of mammalian origin such as from a human, livestock animal (e.g. cow, horse, sheep, goat or donkey), laboratory test animal (e.g. rat, mouse or rabbit), companion animal (e.g. dog or cat) or captive wild animal (e.g. dingo, fox, wild boar or kangaroo).
  • the most preferred receptors are of human and laboratory test animal origin (e.g. murine species, i.e. rat or mouse).
  • antibodies are polyclonal antibodies, they may be generated in any convenient host including a human, livestock animal, companion animal or captive wild animal as hereinbefore described. Where the antibodies are monoclonal antibodies, they may be prepared in any convenient hybridoma such as of murine (e.g. rat or mouse) origin.
  • murine e.g. rat or mouse
  • the receptor used to generate the antibodies may be the whole receptor such as in purified, partially purified or isolated form including in the form of isolated membrane preparations.
  • the receptor may also be produced by recombinant procedures or synthetic procedures or a combination thereof.
  • a fragment of the receptor is used which, in an even more preferred embodiment, is fused to a suitable carrier or marker molecule such as FLAG protein.
  • suitable carrier or marker molecule such as FLAG protein.
  • Alternative carrier molecules include glutathione-S-transferase (GST) or alkaline phosphatase (AP).
  • a molecule interactive with a non-full length G-CSF-R fused to a carrier molecule The non-full length portion of the receptor acts as a hapten.
  • the non-full length receptor comprises its extracellular domain.
  • the carrier molecule is FLAG, AP or GST.
  • the resulting fusion molecule is then used to generate polyclonal or monoclonal antibodies which may undergo immunoadsorbent procedures to provide a composition of substantially, for example, extracellular domain-reactive receptor antibodies.
  • immunointeractive molecules is used herein in its broadest sense and includes antibodies, parts, fragments, derivatives, homologues or analogues thereof, peptide or non-peptide equivalents thereof and fusion molecules between two or more antibodies or between an antibody and another molecule.
  • the antibodies or other immunointeractive molecules may also be in recombinant or synthetic form. Accordingly, the present invention contemplates mutants and derivatives of the immunointeractive molecules, especially when such molecules are antibodies.
  • Mutants and derivatives of such antibodies include amino acid substitutions, deletions and/or additions. Furthermore, amino acids may be replaced by other amino acids having like properties, such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains, interactive and/or functional groups and so on. Glycosylation variants and hybrid antibodies are also contemplated by the present invention. Amino acid substitutions are typically of single residues; insertions will usually be of the order of about 1-10 amino acid residues; and deletions will range from about 1-20 residues. Deletions or insertions preferably are made in adjacent pairs, i.e: a deletion of 2 residues or insertion of 2 residues.
  • amino acid variants referred to above may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations. Techniques for making substitution mutations at predetermined sites in DNA having known sequence are well known, for example through M13 mutagenesis. The manipulation of DNA sequences to produce variant proteins which manifest as substitutional, insertional or deletional variants are well known in the art.
  • recombinant or synthetic mutants and derivatives of the antibodies of the present invention include single or multiple substitutions, deletions and/or additions to any molecule associated with the ligand such as carbohydrates, lipids and/or proteins or polypeptides. Naturally occurring or altered glycosylated forms of the subject antibodies are particularly contemplated by the present invention.
  • Amino acid alterations to the subject polypeptide contemplated herein include insertions such as amino acid and/or carboxyl terminal fusions as well as intra- sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than amino or carboxyl terminal fusions, of the order of about 1 to 4 residues. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Such substitutions may be made in accordance with Table 1. TABLE 1
  • analogues and derivatives also extend to any functional chemical equivalents of the antibodies characterised by their increased stability and/or efficacy in vivo or in vitro.
  • analogue and derivatives further extend to any amino acid derivative of the antibodies as described above.
  • Antibody analogues contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or derivatising the molecules and the use of crosslinkers and other methods which impose conformational constraints on the antibodies.
  • side chain modifications contemplated by the present invention include modifications of amino groups such y reductive alkylation by reaction with an aldehyde followed by reduction wiu.
  • guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3- butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O- acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbomoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate .
  • Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t- butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6- methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • peptides could be conformationally constrained by, for example, incorporation of C a and N legal- methylamino acids, introduction of double bonds between C a and C ⁇ atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
  • the present invention extends to amino acid and/or chemical analogues of the subject antibodies having the identifying characteristics of being interactive with the extracellular domain of the NYK-flk-1 receptor.
  • an antibody includes the naturally occurring molecule, recombinant, synthetic and analogue forms thereof and to any mutants, derivatives and human and non-human homologues thereof including amino acid and glycosylation variants.
  • the immunointeractive molecules of the present invention may be used to develop a new range of therapeutic and diagnostic agents.
  • the antibodies or fragments or derivatives thereof may act as antagonists to disrupt G-CSF and receptor interaction useful which may be useful in the treatment of some tumours and cancers. They may also be used for screening for agonists useful, for example, where G-CSF interaction with its receptor is to be promoted. Normal, abnormal or mutated receptor structure or receptor expression can also be determined through immunoreactivity studies.
  • a method of detecting a G-CSF-R on a cell in a biological sample comprising contacting said sample with an immunointeractive molecule capable of binding to the extracellular domain of said NYK/flk-1 receptor immobilised to a solid support for a time and under conditions sufficient for an immunointeractive molecule-G-CSF-R complex to form and then detecting the presence of said complex.
  • the immunointeractive molecule in G-CSF-R complex is detected by contacting the complex with an antibody against the immunointeractive molecule with the antibody being labelled with a reporter molecule.
  • the immunointeractive molecule itself is labelled with a reporter molecule.
  • the immunointeractive molecules are generally antibodies and these or antibodies directed against the immunointeractive molecules may be polyclonal or monoclonal antibodies and these are obtainable by immunisation of a suitable animal with the immunointeractive molecule (or G-CSF-R) and either type is utilisable in the assay.
  • the methods of obtaining both types of sera are well known in the art.
  • Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of immunointeractive molecule or G-CSF-R preparation, or antigenic parts thereof, collecting s ⁇ rum from the animal, and isolating specific antibodies by any of the known immur ⁇ orbent techniques.
  • antibodies produced by this method are utilisable in virtually any type of assay, they are generally less favoured because of the potential heterogeneity of the product.
  • the use of monoclonal antibodies in the above immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product.
  • the preparation of hybridoma cell lines for monoclonal ibody production derived by fusing an immortal cell line and lymphocytes sensitised against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See, for example Douillard and
  • G-CSF-R receptor may be accomplished in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to US Patent Nos.
  • Sandwich assays are among the most useful and commonly used assays and are particularly useful in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an immunointeractive molecule is brought into contact with a biological sample comprising cells potentially carrying G- CSF-R. After a suitable period of incubation, for a period of time sufficient to allow formation of an immunointeractive molecule-G-CSF-R complex, an antibody specific to the immunointeractive molecule, labelled with a reporter molecule capable of producing a detectable signal, is then added and incubated allowing sufficient time for the formation of a tertiary complex.
  • any unreacted material is washed away, and the presence of the antibody bound to the immunointeractive molecule is determined by observation of a signal produced by the reporter molecule.
  • the results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten.
  • Variations on the forward assay include using an immunointeractive molecule labelled with a reporter molecule.
  • the immunointeractive molecule or cells may be immobilised onto a solid support.
  • Suitable solid supports include glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, poly vinyl chloride or polypropylene.
  • the solid supports may .be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing the molecules to the polymer.
  • reporter molecule is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
  • an enzyme immunoassay an enzyme is conjugated to the immunointeractive molecule or an antibody thereto generally by means of glutaraldehyde or periodate. As will be readily recognised, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change.
  • suitable enzymes include alkaline phosphatase ⁇ nd peroxidase. It is also possible to employ fluorogenic substrates which yield a -luorescent product rather than the chromogenic substrates noted above.
  • the enzyme-labelled antibody is added to the immunointeractive molecule-receptor complex, allowed to bind, and then the excess reagent is washed away.
  • an enzyme labelled immunointeractive molecule is used.
  • a solution containing the appropriate substrate is then added to the tertiary complex.
  • the substrate will react with the enzyme linked to the antibody/immunointeractive molecule, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample.
  • Reporter molecule also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
  • fluorescent compounds such as fluorescein and rhodamine
  • fluorescent compounds may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome- labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome- labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
  • Immunofluoresence and El A techniques are both very well established in the art and are particularly useful for the present method.
  • other reporter molecules such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
  • the present invention also provides a method of screening for G- CSF-R, such as an altered G-CSF-R, by determining the ability of a putative G-CSF- R to interact with an antibody thereto.
  • the method may also be employed to scren for molecules capable of disrupting G-CSF-R antibody interaction thereby providing potential antagonists.
  • the method may also be used to screen for enhanced antibody binding thereby providing potential agaonists.
  • the present invention also provides a pharmaceutical composition comprising an effective amount of an immunointeractive molecule capable of binding or otherwise associating with the extracellular domain of G-CSF-R and one or more pharmaceutically acceptable carriers and/or diluents.
  • the active ingredients of a pharmaceutical composition comprising the immunointeractive molecules are contemplated to exhibit excellent therapeutic activity, for example, in the treatment of angiogenic-dependent phenotype and disease conditions resulting therefrom, such as metastasis, in an amount which depends on the particular case. For example, from about 0.5 ⁇ g to about 20 mg per kilogram of body weight per day may be administered. Dosage procedures may be adjusted to provide the optimum therapeutic response.
  • the active compound may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting (eg using slow release molecules).
  • the active ingredients which comprise the immunointeractive molecules may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredients.
  • they will be coated by, or administered with, a material to prevent its inactivation.
  • the immunointeractive molecules may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes.
  • Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon.
  • Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.
  • Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol.
  • Liposomes include water-in-oil-in- water emulsions as well as conventional liposomes.
  • the active compounds may also be administered parenterally or intraperitoneally.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as licithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • the active, compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 1 % by weight of active compound.
  • compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
  • the amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 10 ⁇ g and 2000 mg of active compound.
  • the tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and formulations.
  • pharmaceutically acceptable carrier and/or diluent includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
  • the principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed.
  • a unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 ⁇ g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 ⁇ g to about 2000 mg/ml of carrier.
  • the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • the immunointeractive molecules used in a pharmaceutical composition are antibodies or mutants or derivatives thereof. Most preferably, the antibodies are monoclonal antibodies.
  • EXAMPLE 1 CHO-6A11 CELLS CHO-K1 cells were transfected by the calcium phosphate method (6) with the pEE6.HCMV.GS expression vector (7,8) carrying a full length cDNA encoding the human G-CSF-R (9). Transfected cells were selected for glutamine synthetase expression by growth in GMEM-S medium (10). Colonies were further tested for expression of the G-CSF-R by determining their ability to bind 125 I-G-CSF. The colony with the highest level of G-CSF binding (CHO-6A11) was selected for further use.
  • FACTOR DEPENDENT CELLS FDC-P1 cells and BAF/BO3 cells were co-transfected by electroporation with the pEFBos expression vector (11) carrying the G-CSF-R cDNA and pGKneo (12). Transfected cells were selected by growth in medium containing 1.2 mg/ml neomycin. Growing colonies were expanded and tested for proliferative response to G-CSF.
  • Rabbits were immunised with a glutathione-S-transferase-G-CSF-R fusion protein (GST-GR) containing the haemopoietin receptor domain.
  • GST-GR glutathione-S-transferase-G-CSF-R fusion protein
  • a BamHl fragment of the receptor cDNA encoding amino acids 17-345 was sub-cloned into the BamHl site of pGEX-1 (13) and the construct was expressed in E. coli.
  • the fusion protein was partially purified by affinity chromatography as described (13).
  • Rabbits were injected with 100 ⁇ g GST-GR emulsified in complete Freund's adjuvant subcutaneously. After 6 weeks, rabbits were boosted with 100 ⁇ g GST-GR in incomplete Freund's adjuvant then bled after a further 2 weeks. The best antiserum was designated R55.
  • mice were immunised with about 2 x 10 7 CHO-6A11 eells intraperitoneally in PBS. The mice were boosted after 2 and 4 weeks with the same inoculum. At least one month after the last boost, mice were given a final boost I/P and their spleens were fused 4 days later with P3-NSl/l-Ag4-ll murine myeloma cells by standard methods (14).
  • Glutaraldehy de (0.05% v/v, 200 ⁇ l/well) was added and the cells allowed to fix for 10 min at room temperature. Plates were then washed with PBS containing 0.02% v/v Tween 20 four times and stored in PBS at 4°C until required. Using 0.05% w/v glutaraldehy de allowed all the antibodies tested to recognise the CHO-6A11 cells; higher concentrations destroyed some epitopes. Antibody binding was detected with biotinylated anti- ⁇ chain antibody (Amersham) followed by avidin-biotinylated peroxidase complex (Dako). Antibodies that were positive in this assay were also tested by fluorescence.
  • Hybridoma culture supernatant was precipitated with 50% V/V saturated ammonium sulphate at 4°C and the precipitate was dissolved in deionised water then dialysed against PBS.
  • IgG antibodies were further purified by Protein A-Sepharose affinity chromatography.
  • EXAMPLE 6 FLUORESCENCE ANALYSIS
  • CHO-6A11 and CHO- 10 cells were harvested using PBS containing 2 MM EDTA and resuspended in PBS containing 10% v/v fetal calf serum (PBS-FCS). Monoclonal antibodies were added at a final concentration of 10 ⁇ g/ml and incubated for 30 min at 4°C. Cells were washed and resuspended in FITC-conjugated sheep anti-mouse Ig (Silenus) diluted 1: 100 in PBS-FCS. Samples were incubated for 30 min at 4°C then washed twice.
  • CHO-6A11 cells were lysed in lysis buffer (50 mM Tris/HCl, 150 mM NaCl, 1 % v/v Triton-X-100, 1 mM EDTA and protease inhibitors) and the protein concentration of the lysate was determined by Bradford assay (Biorad) using bovine serum albumin as a standard. Aliquots of cell lysate were pre-cleared with rabbit anti-mouse immunoglobulin (Dakopatts) and protein A-Sepharose (Pharmacia) for 2 hr at 4°C.
  • lysis buffer 50 mM Tris/HCl, 150 mM NaCl, 1 % v/v Triton-X-100, 1 mM EDTA and protease inhibitors
  • the cleared lysates were incubated with monoclonal antibodies (3 ⁇ g/ml purified antibody or 20 ⁇ l/ml of hybridoma supernatant) for 2 hr at 4°C, then complexes were precipitated with rabbit anti-mouse immunoglobulin and protein A-Sepharose. Immunoprecipitates were analysed by SDS-polyacrylamide electrophoresis (PAGE), reducing conditions, on a 6% w/v acrylamide gel. Protein was then electrophoretically transferred to nitrocellulose and blocked overnight at 4°C with 10% w/v skim milk powder solution.
  • the nitrocellulose was incubated with rabbit anti-G-CSF-R antibody purified on protein A-Sepharose (R55, 0.4 ⁇ g/ml) followed by peroxidaseconjugated swine anti-rabbit immunoglobulin (1:1000, Dakopatts) and enhanced chemiluminescence (ECL) (Amersham).
  • rabbit anti-G-CSF-R antibody purified on protein A-Sepharose (R55, 0.4 ⁇ g/ml) followed by peroxidaseconjugated swine anti-rabbit immunoglobulin (1:1000, Dakopatts) and enhanced chemiluminescence (ECL) (Amersham).
  • the receptor was immunoprecipitated with 15 ⁇ l of a 50% slurry of Sepharose-conjugated LMM741 (5 mg antibody / ml Sepharose) and blotted with monoclonal anti-receptor antibodies (0.5 ⁇ g/ml) followed by peroxidase-conjugated rabbit anti-mouse immunoglobulin (1: 1000, Dakopatts) and ECL.
  • FD.hGR3 cells or BAG.hGR cells were washed 3 times and resuspended in DME medium with 10% v/v heat inactivated fetal calf serum at 5 x lOVml (FD.hGR) or 1 x K ⁇ /ml (BAF.hGR).
  • Monoclonal antibodies were diluted to 400 ⁇ g/ml in medium and usually 2-fold serial dilutions were performed.
  • Recombinant hG-CSF (Amgen) was diluted to 2 ng/ml in medium. Fifty ⁇ l of antibody dilution, 50 ⁇ l of G-CSF and 100 ⁇ l of cell suspension were added to wells of 96- well tissue culture plates (Nunc) in triplicates.
  • Plates were incubated for 24 hr at 37°C. After 20 hr or 44 hr, respectively, of incubation, plates were pulsed with 0.5 ⁇ Ci/well 3 H-thymidine for 4 hr then harvested onto glass fibre filter paper and counted in an LKB Betaplate liquid scintillation counter.
  • YPY-rh-G-CSF (Tyr 1,3-G-CSF) (Kirin) was iodinated by the lactoperoxidase-glucose oxidase method (15), giving a specific activity of 0.9-3 x 10 8 cpm/ ⁇ g, as determined by self displacement analysis (16) or ELISA.
  • BAF.hGR cells were cultured overnight in medium containing WEHI-3B D " conditioned medium instead of G-CSF before use.
  • Antibodies to be tested wre diluted in RPMI medium containing 10 mM HEPES buffer and 10% v/v FCS (RHF).
  • the cells were washed once with RPMI, resuspended in cold RHF and diluted to 3.3 x 10 6 /ml.
  • Antibody dilutions were added (50 ⁇ l/tube) to 1.5 ml tubes followed by 100 ⁇ l of RHF or unlabelled rhG-CSF (3 ⁇ g/ml, 50 ⁇ l of 125 I-G-CSF (approximately 40,000 cpm) and

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Abstract

L'invention porte sur des molécules interactives de récepteurs de cytokine et leur utilisation dans la mise au point de nouvelles molécules à usage thérapeutique et diagnostique. L'invention porte plus particulièrement sur des molécules immunoréactives, telles que des anticorps, capables d'interagir avec le récepteur du facteur GSF.
EP95911668A 1994-02-08 1995-02-08 Molecules interactives du recepteur du facteur de stimulation des colonies de granulocytes Withdrawn EP0807123A4 (fr)

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AUPM3751A AUPM375194A0 (en) 1994-02-08 1994-02-08 Cytokine receptor interactive molecules
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AUPM375094A0 (en) * 1994-02-08 1994-03-03 Ludwig Institute For Cancer Research Cytokine receptor interactive molecules and methods for assaying cytokine function
US20020009724A1 (en) * 1999-12-08 2002-01-24 Robert Schlegel Compositions, kits, and methods for identification, assessment, prevention, and therapy of cervical cancer
JP4611738B2 (ja) * 2002-08-23 2011-01-12 ザ ウォルター アンド エリザ ホール インスティテュート オブ メディカル リサーチ 治療および予防の方法
AU2012269720B2 (en) * 2011-06-13 2015-01-22 Csl Limited Antibodies against G-CSFR and uses thereof

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WO1995021864A1 (fr) * 1994-02-08 1995-08-17 Ludwig Institute For Cancer Research Anticorps de fixant au domaine extracellulaire du recepteur du facteur de croissance des colonies de granulocytes g-csf et procedes de traitement

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US4683295A (en) * 1984-05-24 1987-07-28 Scripps Clinic And Research Foundation Method for the preparation of anti-receptor antibodies
ATE123812T1 (de) * 1989-09-26 1995-06-15 Immunex Corp Rezeptoren für granulozytenkolonien stimulierenden faktor.

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WO1995021864A1 (fr) * 1994-02-08 1995-08-17 Ludwig Institute For Cancer Research Anticorps de fixant au domaine extracellulaire du recepteur du facteur de croissance des colonies de granulocytes g-csf et procedes de traitement

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
A. SUREDA ET AL.: "Analysis of granulocyte colony stimulating factor receptor expression on human cell lines and murine cells by means of a purified mAb." TISSUE ANTIGENS, vol. 42, no. 4, 1993, page 337 XP002122100 Copenhagen, Denmark *
J. LAYTON ET AL.: "The use of monoclonal antibodies and alanine substitution mutants to define the G-CSF receptor-binding site." LYMPHOKINE AND CYTOKINE RESEARCH, vol. 12, no. 5, 1993, page 327 XP002122102 New York, NY, USA *
J. LI ET AL.: "Growth-stimulation and differentiation-promotion mediated by granulocyte colony-stimulating factor receptor in different hematopoietic cell lines." PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, vol. 35, March 1994 (1994-03), page 565 XP002122103 USA *
See also references of WO9521867A1 *
V. BROUDY ET AL.: "Monoclonal antibody 4B10 (M33) recognizes stem cell factor (SCF), and antibody VIMD2b (M16) recognize the receptor for granulocyte colony stimulating factor (G-CSF)." TISSUE ANTIGENS, vol. 42, no. 4, 1993, page 331 XP002122101 Copenhagen, Denmark *

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EP1156063A1 (fr) 2001-11-21

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